The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

Abstract

The epsins are a family of adaptors involved in recruiting other endocytic proteins, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. We have previously demonstrated that the ENTH domain of epsin is involved in Cdc42 signaling regulation. Here, we present evidence that yeast epsin 2 (Ent2) plays a signaling role during cell division. We observed that overexpression of the ENTH domain of Ent2 (ENTH2), but not Ent1, promoted the formation of chains of cells and aberrant septa. This dominant-negative effect resulted from ENTH2-mediated interference with septin assembly pathways. We mapped the ENTH2 determinants responsible for induction of the phenotype and found them to be important for efficient binding to the septin regulatory protein, Bem3. Supporting a physiological role for epsin 2 in cell division, the protein localized to sites of polarized growth and cytokinesis and rescued a defect in cell division induced by Bem3 misregulation. Collectively, our findings provide a potential molecular mechanism linking endocytosis (via epsin 2) with signaling pathways regulating cell division.

Bem3, a Cdc42 GTPase-Activating Protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

“…We also showed that cell division defects caused by overexpression of a hyperactive Bem3 mutant (Bem3 D1-114 ) were suppressed by co-overexpression of the endocytic protein, Ent2 (Mukherjee et al, 2009). Further supporting the role of vesicle trafficking in Bem3 localization, Bem3 has been reported to localize to an unidentified intracellular compartment (Knaus et al, 2007) and actin cables have been shown to play an essential role in its transport to polar sites (Knaus et al, 2007).…”

“…We have previously reported that cell division defects resulting from overexpression of the constitutively active Bem3 mutant (Bem3 D1-114 ) in yeast (Kadota et al, 2004) are rescued by cooverexpression of the endocytic protein Ent2 (Mukherjee et al, 2009). Therefore, we hypothesized that vesicle trafficking, including endocytosis, plays an important role in the regulation of Bem3 activity.…”

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis

“…But we note that GFP-Ent1 and GFPEnt2 were previously shown to complement an ent1D ent2D mutant, so it seems unlikely that their differences are caused solely by the tag (Watson et al, 2001). Moreover, other studies have shown that the two endocytic epsins have functions that are not completely overlapping (Baggett et al, 2003;MaldonadoBáez et al, 2008;Mukherjee et al, 2009;Newpher et al, 2005;Wendland et al, 1999). Further studies will be needed to explain the differences in adaptor behavior during endocytosis.…”

RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking

“…79 This observation, along with the involvement of the RE and the exocyst, further expand the similarities with the cases of Bem3/Ocrl1. Indeed, Bem3 42 and Ocrl1 (Aguilar lab, unpublished results) are epsin-interacting partners as well. Collectively, these studies indicate conservation of the functions of the small GTPase binding proteins in polarized membrane traffic.…”

“…41 Not surprisingly, Bem3 colocalizes with septins both at the polar cap prior to bud emergence, and at the bud neck during cytokinesis. 40,42 A recent study from our lab established that Bem3 also localizes to an intracellular compartment that is distinctly visible during early stages of the cell cycle. 37 Indeed, electron micrographs revealed that Bem3 occupies a cluster of vesicles polarized toward the emerging bud ( Fig.…”